During the development of neuronal circuits, axons and their collateral branches are guided by attractive and repulsive cues to postsynaptic targets. The guidance cue Netrin-1 and its receptor Deleted in Colorectal Cancer (DCC) are well-established factors in axonal guidance and branching. However, the mechanisms downstream of Netrin-1:DCC that control axon branching and guidance are not known. We recently identified TRIM9, an E3 ubiquitin ligase expressed exclusively in the nervous system, as a critical downstream component of netrin signaling. This proposal will test the hypothesis that TRIM9 acts as a master regulator of netrin dependent axon branching and guidance through its direct interaction with the netrin receptor DCC. The first aim will investigate the importance of TRIM9 in DCC localization and examine the critical role TRIM9 plays in the regulation of signaling downstream of Netrin/DCC via the Focal Adhesion Kinase (FAK) pathway. The second aim will attempt to rescue the aberrant thickening of the corpus callosum found in TRIM9-/- mice through the genetic inhibition of FAK, and determine whether loss of TRIM9 promotes the misprojection of cortical axons in vivo. This proposal combines innovative TIRF microscopy, innovative cell biological approaches, and neuroanatomical investigations in order to illuminate key regulators of proper axon branching and guidance.